The Flaviviridae are a diverse family of enveloped viruses infecting both arthropods and vertebrates. Flaviviruses have a positive-sense single-stranded RNA genome 10.7 kb in length, transcribed into a single polyprotein precursor encoding three structural proteins, capsid, premembrane (prM), envelope (E), and seven non-structural proteins (Lindenbach & Rice, Flaviviridae: the viruses and their replication. In Fields Virology, 4th ed., Knipe and Howley. Eds., Philadelphia, Lippincott Williams & Wilkins, pp. 991-1041, 2001; Rice et al., Science 229:726-33, 1985). The flavivirus E-glycoprotein is the primary antigen, inducing protective immunity; it is essential for membrane fusion, and mediates binding to cellular receptors (Allison et al., J. Virol. 75:4268-75, 2001; Crill & Roehrig, J. Virol. 75:7769-73, 2001; Rey et al., Nature 375:291-98, 1995). Flavivirus E-glycoprotein therefore directly affects host range, tissue tropism, and the virulence of these viruses.
The flavivirus E-glycoprotein contains three structural and functional domains. Domain I (DI) is an 8-stranded β-barrel containing two large insertion loops that form the elongated finger-like domain II (DII) (Rey et al., Nature 375:291-98, 1995). DII is involved in stabilizing the E-glycoprotein dimer and contains the internal fusion peptide (Allison et al., J. Virol. 75:4268-75, 2001). Domain III (DIII) forms a ten-stranded β-barrel with an immunoglobulin-like fold and contains the cellular receptor-binding motifs (Crill & Roehrig, J. Virol. 75:7769-73, 2001; Modis et al., PNAS 100:6986-91, 2003). DI and DIII contain predominately type-specific and subcomplex-reactive epitopes, whereas DII contains the major flavivirus group- and subgroup-cross-reactive epitopes, which are sensitive to reduction and denaturation and are formed from discontinuous amino acid sequences (Mandl et al., J. Virol. 63:564-71, 1989; Rey et al., Nature 375:291-98, 1995; Roehrig et al., Virology 246:317-28, 1998).
Members of the Flaviviridae family that infect humans frequently cause severe morbidity and mortality, and epidemics of flaviviruses continue to be a major public health concern worldwide. More than two billion people are at risk of being infected with members of the genus Flavivirus which includes at least 70 distinct virus species (Burke & Monath, Flaviviruses. In Fields Virology, 4th ed., Knipe and Howley. Eds., Philadelphia, Lippincott Williams & Wilkins, pp. 1043-1125, 2001; Kuno et al., J. Virol. 72:73-83, 1998; Solomon & Mallewa, J. Infect. 42:104-15, 2001). The medically important flaviviruses include yellow fever (YF) virus in Africa, Latin and South America; Japanese encephalitis (JE) virus in Asia and Australia; West Nile (WN) virus in Africa, Central Europe, and most recently in North America; tick-borne encephalitis (TBE) complex viruses in the temperate regions of Europe, North America and Asia; and the four serotypes of dengue viruses (DEN-1, -2, -3, and -4) in tropical and subtropical regions of the world (Lindenbach & Rice, Flaviviridae: the viruses and their replication. In Fields Virology, 4th ed., Knipe and Howley. Eds., Philadelphia, Lippincott Williams & Wilkins, pp. 991-1041, 2001).
Human infection by flaviviruses results in a humoral immune response involving virus species-specific as well as flavivirus cross-reactive antibodies (Calisher et al., J. Gen. Virol. 70:37-43, 1989; Tesh et al., Emerg. Inf. Dis. 8:245-51, 2002). The presence of flavivirus cross-reactive antibodies in human sera produces two public health concerns upon secondary infection with a heterologous flavivirus. Serodiagnosis of secondary flavivirus infections, especially in areas with multiple co-circulating flaviviruses, can be particularly difficult due to the inability to differentiate primary from secondary cross-reactive serum antibodies using currently available viral antigens. Therefore, definitive epidemiological information either cannot be obtained or is delayed to the point that effective control and prevention strategies may be delayed. Additionally, the presence of sub-neutralizing levels of flavivirus cross-reactive serum antibodies may result in increasing the severity of secondary flavivirus infections due to antibody-dependent enhancement (ADE), in particular, following secondary dengue virus infection (Ferguson et al., PNAS 96:790-94, 1999; Halstead, Rev. Infect. Dis. 11:830-39, 1989; Takada & Kawaoka, Rev. Med. Virol. 13:387-98, 2003; Wallace et al., J. Gen Virol. 84:1723-28, 2003). Thus, there exists a need for a method for identifying and characterizing flavivirus cross-reactive epitopes for improved flavivirus serodiagnosis and development of flavivirus vaccines.